This application relates to the isolation of cell free extracts from a microorganism selected from the family Tetrahymenida, such as Tetrahymena thermophila. One aspect of the present invention is the provision of methods for the conversion of cholesterol present in foodstuffs into cholesterol desaturated derivatives, including provitamin D, using the cell free extracts.
Animal milk is a complex mixture of different compounds, including lipids, proteins, minerals, sugars and vitamins (Russof, L. L. (1970). J. Dairy Science 53:1296-1302). The calcium, phosphate and vitamin D content of milk make it an adequate source of nutrients for bone formation (Fox, P. F. and McSweeney, P. L. H. (1998a) Salts of milk. In xe2x80x9cDairy Chemistry and Biochemistryxe2x80x9d, chapter 5, Blackie Academic and Professional, London). This may be a key aspect of its role in nature, allowing mammalian newborns to complete the formation of the skeleton after birth. Mineral and vitamin components of milk are also important to preserve bone structure in adulthood. Milk is also relatively economical, compared to other animal protein sources, and thus it makes a valuable contribution to the human diet (Russof, L. L. (1970) J. Dairy Science 53:1296-1302).
The lipid fraction of milk includes cholesterol, however, which has been implicated as a causative agent of coronary artery disease (Artaud-Wild, S. M., Connor, S. L., Sexton, G., Connor, W. E. (1993) Circulation 88: 2771-2779). In effect, the increased blood cholesterol concentration in humans seems to have a direct positive correlation with coronary heart disease. Therefore patients with coronary heart disease (CHD) or hypercholesterolemia are commonly recommended to decrease their dietary cholesterol intake.
Other foodstuffs of animal origin such as eggs, which are commonly used in the preparation of a variety of food products, present the same problem. Because of the special organoleptic traits of milk and eggs, it is difficult to replace them by other products with less cholesterol content.
The general awareness of the risks associated with high blood cholesterol levels is an important factor limiting the consumption of food substances that have high cholesterol content by a health-conscious public. In the past years it has been a significant health trend away from red meat, milk and eggs. Accordingly, there is a continuing and real interest in decreasing the intake of food substances that have high cholesterol content.
To address these problems, there is a need for methods to produce low-cholesterol versions of normally high-cholesterol foodstuffs, such as whole milk and eggs. Such methods should preferably not appreciably change the physical and organoleptic properties of the foodstuffs. The nutritional value of the treated foodstuffs should be preferably maintained, especially the levels of those components that are lipid-soluble and that are important for human nutrition (e.g., vitamins A and D, and essential fatty acids). Thus, the food treatment methods should yield products with lower cholesterol content but which are otherwise similar to the untreated foodstuffs. Additionally, the novel methods should preferably not require expensive equipment and materials or potentially toxic materials, such as organic solvents.
A number of methods have been described in patents in the US and other countries for reducing the cholesterol content of foodstuffs. For example, cholesterol can be removed from foodstuffs by the use of physicochemical methods. For instance, the use of supercritical fluids to produce liquid egg having reduced cholesterol content has been proposed (Ogasahara et al, U.S. Pat. No. 5,116,628, 1992). However, the high temperatures and pressures needed for the process can denature proteins present in the foodstuffs. Likewise, the production of low cholesterol butter oil by vapor sparging (Conte et al, 1992, U.S. Pat. No. 5,092,964) is another example of a method, which, due to the extreme conditions used, is likely to denature proteins and alter organoleptic properties of the foodstuffs.
The use of organic solvents to extract cholesterol from foodstuffs has also been proposed. Thus, Fallis et al (1978, U.S. Pat. No. 4,104,286) have proposed the use of aqueous ethanol, saponification, and extraction with hydrocarbons and methanol to obtain free cholesterol, saponified fats and edible egg powder. This process uses extreme conditions and large quantities of organic solvents that may contaminate the processed foodstuffs. Extraction with liquid dimethylether (Yano et al, 1980, U.S. Pat. No. 4,234,619) is similarly inconvenient and does not appear to be selective for cholesterol as other neutral lipids are removed from the foodstuff. Johnson et al. (1991, U.S. Pat. No. 4,997,668) applied solvent extraction to milk, but again the method does not appear to be selective for cholesterol and utilizes organic solvents that may contaminate foodstuffs.
A variation on the use of organic solvents is to employ oils to extract cholesterol from either aqueous or dry foodstuffs, like egg yolk and dairy products. (Bracco et al, 1982, U.S. Pat. No. 4,333,959; Keen, 1991, U.S. Pat. No. 5,039,541; Conte et al, 1992, U.S. Pat. No. 5,091,203; Merchant et al, 1995, U.S. Pat. No. 5,378,487; Jackeschky, 1998, U.S. Pat. No. 5,780,095). Again, these methods do not selectively extract cholesterol and oils contaminated with cholesterol are inevitably produced, which is undesirable.
Removal of cholesterol by formation of complexes with cyclodextrins has also been proposed for fatty substances of animal origin (Courregelongue et al, 1989, U.S. Pat. No. 4,880,573) and specifically in the case of dairy products (Chung Dae-Won, 1999, Foreign Patent WO 9917620). The formation of complexes of cholesterol and saponin has also been described as a means to reduce cholesterol in milk (Richardson, 1994, U.S. Pat. No. 5,326,579). These methods are, however, too expensive for industrial applications.
A different approach is based on the use of enzymes that modify cholesterol. Thus, the use of cholesterol reductases, that modify cholesterol into poorly absorbed sterols, has been proposed (Beitz et al, 1990, U.S. Pat. No. 4,921,710; Ambrosius et al, 1999, U.S. Pat. No. 5,856,156). Another proposed enzymatic approach is the conversion of cholesterol into epicholesterol, which is then further modified by an epicholesterol dehydrogenase (Saito et al, 1999, U.S. Pat. No. 5,876,993). These methods have the disadvantage that they do not result in the conversion of cholesterol into useful compounds for human nutrition.
There is therefore a need for methods for treating foodstuffs to reduce the amount of cholesterol. Preferably, the cholesterol is converted to one or more substances that are useful for human nutrition.
When Tetrahymena is grown in the presence of exogenous sterols, the biosynthesis of tetrahymanol is completely inhibited and the added sterol is accumulated by the organism and, in most cases, metabolized to other sterols. Different types of biotransformations have been observed, including xcex947 and xcex9422 desaturation and the removal of ethyl, but not methyl groups, from C24 (Mallory, F. B. and Conner, R. L. (1971) Lipids 6:149-153; Conner, R. L., Landrey, J. R., Joseph, J. M., Nes, W. R. (1978) Lipids 13: 692-696; Ferguson, K. A., Davis, F. M., Conner, R. L., Landrey, J. R. and Mallory, F. B. (1975) J Biol Chem 250:6998-7005).
In the case of cholesterol, Tetrahymena transforms this sterol into the desaturated derivatives: xcex947,22-bis-dehydrocholesterol (a close analog of ergosterol, also called provitamin D2), xcex9422-dehydrocholesterol and xcex947-dehydrocholesterol (provitamin D3). (Conner, R. L., Mallory, F. B., Landrey, J. R. and Iyengar, C. W. L. (1969), J Biol Chem 244:2325-2333). The desaturation of cholesterol at position 7 converts it into provitamin D3 and pro vit D derivatives, which upon UV irradiation in the skin can be activated to vitamin D. This biotransformation has seldom been observed in nature.
The present invention describes the isolation of cholesterol desaturases, including xcex947, xcex9422 and xcex947, 22 desaturases, in cell free extracts from Tetrahymena.
It is believed that the applicants are the first ever to isolate this activity in cell free extracts and to use these fractions for treating cholesterol-containing foodstuffs.
The cell free extract can be used in the processing of animal food products, reducing cholesterol while simultaneously enhancing provitamin D availability.
The present invention utilizes the foregoing properties of the cell free extract from Tetrahymena, and other protozoa, to provide methods for treating foodstuffs (such as milk, egg and other foodstuffs) to achieve the following goals: (a) reduce the level of cholesterol in foodstuffs and (b) increase the level of cholesterol desaturated derivatives.
It is a primary object of the present invention is to provide methods for reducing the amount of cholesterol in cholesterol-containing foodstuffs, using cell free extracts from Tetrahymena, preferably without significantly altering the organoleptic properties of the treated foodstuffs.
It is a further object of the present invention to provide means of decreasing the amount of cholesterol in dairy products, such as milk, egg and other products of animal origin.
It is another object of the present invention to provide methods for increasing the amount of xcex947-dehydrocholesterol, xcex9422-dehydrocholesterol and xcex947-22didehydrocholesterolin foodstuffs. The present invention provide a method for increasing the amount of provitamin D in foodstuffs. Examples of vitamin D precursors are: xcex947-dehydrocholesterol (provitamin D3) and xcex947-22didehydrocholesterol(a close analog of ergosterol, provitamin D2)
In accordance with the foregoing, in one aspect the present invention provides methods for preparing cell free extracts of Tetrahymena, including homogenate, microsomal fraction and desaturase-enriched preparation for treating cholesterol containing foodstuffs.
In another aspect the present invention provides methods for changing of sterols profiles of a foodstuff, said methods comprising at least the step of treating a cholesterol-containing foodstuff by incubate the same with a cell free extract obtained from a Tetrahymenidae family microorganism in a effective amount to enable the following changes in the composition of the foodstuff: (a)reduce the level of cholesterol and (b) increase the level of at least one cholesterol desaturated derivative: xcex947-dehydrocholesterol, xcex9422-dehydrocholesterol or xcex947-22 bisdehydrocholesterol.
Any foodstuff of animal origin can be treated in accordance with the methods of the present invention . The foodstuff can be a dairy product, such as milk, egg yolk or an egg product. The milk can be milk from any mammal, such as cow or goat, and egg yolk can be from eggs of any species of bird, such as domestic chicken eggs. Representative examples of other foodstuffs that can be treated by the methods of the present invention are broth, yogurt, cheese, cream and soups that contain material of animal origin including cholesterol.
The cell free extract comprise at least one cholesterol desaturase that catalyze desaturation of cholesterol to a more desaturated derivate (e.g. xcex947-dehydrocholesterol, xcex9422-dehydrocholesterol or xcex947-22 bisdehydrocholesterol. The cell free extract can be an homogenate, a microsomal fraction or an enriched-desaturase fraction obtained from any Tetrahymenidae family microorganism.
When the methods of the present invention are utilized to reduce the level of cholesterol in milk, the level of cholesterol in the milk after treatment is preferably less than 90%, more preferably less than 70%, and more preferably less than 30% of the level of cholesterol in the milk before treatment.
In one embodiment of the invention the homogenate traction obtained from the culture of a member of the family Tetrahymenidae is incubated under conditions that enable a reduction in the level of cholesterol in the treated foodstuff and an increase in the level of at least one cholesterol desaturated derivative, such as xcex9422-dehydrocholesterol or a vitamin D precursor (formed by the biochemical conversion of the cholesterol). Representative examples of vitamin D precursors are xcex947-dehydrocholesterol and xcex947 22-didehydrocholesterol.
In another embodiment of the invention a microsomal fraction obtained from cell free homogenates is incubated under conditions that enable a reduction in the level of cholesterol in the treated foodstuff and an increase in the level of at least one cholesterol desaturated derivative, (formed by the biochemical conversion of the cholesterol in the treated foodstuff) such as xcex9422-dehydrocholesterol or at least one vitamin D precursors, xcex947-dehydrocholesterol or xcex947 22-didehydrocholesterol.
In another embodiment of the invention a desaturase-enriched fraction obtained from cell free homogenates by extraction and purification is incubated under conditions that enable a reduction in the level of cholesterol in the treated foodstuff and an increase in the level of at least one cholesterol desaturated derivative, (formed by the biochemical conversion of the cholesterol in the treated foodstuff), such as xcex9422- dehydrocholesterol or at least one vitamin D precursors, xcex947-dehydrocholesterol or xcex947 22-didehydrocholesterol
Representative incubation conditions for lowering the level of cholesterol and increasing the cholesterol desaturated derivatives in liquid suspensions such as milk or diluted egg yolk utilizing Tetrahymena termophila cell free extract are: incubation temperature of from 24xc2x0 C., to 37xc2x0 C., at a pH of between 5.0 and 8.0, for a time period of from 1 hour to 24 hours.
For example, the methods of the present invention can be used to decrease between 10% to 90% the level of cholesterol initially present in foodstuff to more desaturated cholesterol derivatives such as: xcex947-dehydrocholesterol, xcex9422-dehydrocholesterol and xcex947-22 bisdehydrocholesterol. Typically, about 90% of the cholesterol originally present in foodstuff is recovered as cholesterol or desaturated cholesterol derived after treatment in accordance with the present invention.
The methods of the present invention provide several advantages. For example, the methods not only selectively decrease foodstuff cholesterol concentration, preferably without significantly altering organoleptic properties, but at the same time, the process of desaturating cholesterol at position 7, converts it into xcex947-dehydrocholesterol, also known as provitamin D3. Thus, a single reaction decreases an unwanted material (cholesterol), converting it into a desirable one (provitamin D3).
The methods of the present invention make unnecessary milk supplementation with vitamin D by providing provitamin D in amounts that are sufficient for daily requirements. In addition, while vitamin D (a supplement normally added to milk), can have toxic effects, the provitamins have a much lower risk of toxicity. This is due to the fact that the provitamins lack vitamin D activity and they have to undergo chemical conversion by exposure to UV light to become active vitamins.
The methods of the present invention avoid the addition of living organisms therefore reducing the risks of undesirable effects.
In another aspect the present invention provides a cell free extract from Tetrahymenidae family microorganism, wherein said cell free extract containing cholesterol desaturase activities such as xcex94-7 and xcex94-22 cholesterol desaturases activities that catalyze desaturation of cholesterol, said cell free extract can be a homogenate fraction, microsomal fraction and desaturase-enriched fraction from Tetrahymenidae family microorganism.
In another embodiment the present invention provided a product, such as liquid egg yolk, dehydrated egg yolk, milk, skimmed milk, yogur, cream, cheese, dehydrated milk, obtained by the method of the invention.